Previous research has indicated that facial attractiveness may provide cues to the functioning of the immune system. Mating with individuals who have a more effective immune system could lead to a higher reproductive success. Our main aim was to test a possible association between immunoreactivity (stimulated by vaccination) and perceived facial attractiveness and healthiness. We experimentally activated the immune system of healthy men using vaccination against hepatitis A/B and meningococcus and measured levels of specific antibodies (markers of immune system reactivity) before and 30 days after the vaccination. Further, 1 day before the vaccination, we collected their facial photographs that were judged by females for attractiveness, healthiness, and facial skin patches for healthiness. In view of its proposed connection with the functioning of the immune system, we also measured skin colouration  and we assessed its role in attractiveness and healthiness judgements. Moreover, we measured the levels of steroid hormones (testosterone and cortisol) and the percentage of adipose tissue, because both are known to have immunomodulatory properties and are related to perceived facial attractiveness and healthiness. We found no significant associations between antibody levels induced by vaccination and perceived facial attractiveness, facial healthiness, or skin healthiness. We also found no significant connections between steroid hormone levels, the amount of adipose tissue, rated characteristics, and antibody levels, except for a small negative effect of cortisol levels on perceived facial healthiness. Higher forehead redness was perceived as less attractive and less healthy and higher cheek patch redness was perceived as less healthy, but no significant association was found between antibody levels and facial colouration. Overall, our results suggest that perceived facial attractiveness, healthiness, and skin patch healthiness provide limited cues to immunoreactivity, and perceived characteristics seem to be related only to cortisol levels and facial colouration. It is often claimed that facial attractiveness provides cues to various aspects of individuals’ quality, such as immunocompetence3. Selection of partners with a more effective immune system is expected to lead to a higher reproductive success by passing increased pathogen resistance onto the offspring (indirect benefits). Moreover, healthier individuals can provide better parental care and are less likely to transmit any infections to their partners (direct benefits)4.Mate preferences are often based on physical appearance, whereby facial attractiveness seems to play an especially significant role6. Several recent studies employed direct immunity function measures, such as inflammation markers or levels of cytokines or antibodies. In a sample of South African men, Phalane et al.7 tested the relationship between facial attractiveness ratings and responsiveness of the immune system upon activation by an injection of bacterial lipopolysaccharide (LPS). Immune system response was assessed by levels of C-reactive protein, which is an inflammation marker, and by the levels of cytokines, which are peptides that stimulate the immune response. This study found a positive correlation between facial attractiveness ratings and the levels of cytokines, specifically interleukins (IL)-2, 4, 6, 8, and 10, granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon γ (IFN-γ), and tumour necrosis factor α (TFN-α)7. Other studies employed vaccinations to elicit and measure immune system reactivity. A stronger response to the vaccine (assessed via higher antibody levels) indicates a better protection against infection8. Overall, though, the results of these studies are inconclusive. Faces of men with higher levels of hepatitis B antibodies were rated as more attractive9 but this did not hold of women10. In contrast, other study reported a negative, though nonsignificant, association between facial attractiveness and immune system reactivity in men11.Previous research into the putative relationship between facial attractiveness and individual's quality that was conducted using self-reported past and current health and attractiveness ratings of facial photographs delivered mixed results15. Studies tend to focus on facial skin colouration in the CIE L*a*b* colour space17. Higher skin redness (a*) is linked to increased skin blood perfusion and oxygenation16, which are in turn positively associated with physical fitness19 as well as good cardiovascular20 and pulmonary health18. Skin yellowness (b*) is influenced by carotenoids, which are pigments acquired from food, mainly fruits and vegetables. Owing to their antioxidant properties21, carotenoids can contribute to disease resistance as they can destroy free radicals and reduce oxidative stress, both of which are harmful to the immune system23. It has been shown that facial skin with higher redness and yellowness is perceived as more attractive and healthier24. Moreover, Phalane et al.7 reported an association between skin yellowness and a marginally higher immune system response (higher levels of inspected cytokines) after LPS stimulation. On the other hand, Foo et al.25 found that higher skin yellowness is positively associated only with perceived health (and only in men) and not with direct immune function measures25. Skin lightness (L*) is determined by the distribution pattern of melanosomes in keratinocytes and the amount of melanin it contains26. Higher melanin levels (resulting in a darker skin hue) can provide a better protection against sunlight27 but can also contribute to vitamin D deficiency28. It has been found that melatonin can have an effect on the synthesis of melanin29, which is in turn believed to affect the periodicity of immune response as well as cytokine production31. In women, lighter skin is associated with higher perceived attractiveness and youth34 , because with increasing age skin tends to become darker36. In men, some research shows that darker complexion may be preferred37.It has been suggested that facial skin colouration plays an important role in perceived facial attractiveness and health40 but evidence to that effect is rather mixed41. It has thus been proposed that glucocorticoids, such as cortisol, mediate the association between testosterone and the immune system functioning43. Although a short-term elevation of cortisol levels can boost an acute immune system response, prolonged exposure may weaken the response, thereby increasing susceptibility to diseases44. Some support for the mediating effect of cortisol comes from Rantala et al.9 who found that immunoreactivity was stronger in men with higher testosterone and simultaneously lower cortisol levels, while immunoreactivity was also positively linked to facial attractiveness. Similarly, women with lower cortisol levels were perceived as more attractive45 .The association between functioning of the immune system and perceived facial attractiveness might be also modulated by testosterone and cortisol. It has been suggested that testosterone has an immunosuppressive effect50. In perception studies, body fat levels affect attractiveness ratings, whereby both overweight and excessively thin individuals are perceived as less attractive51. Moreover, portrait photographs of individuals with elevated levels of leptin—a hormone produced by the adipose tissue that has a negative effect on health—were also perceived as less attractive52.Another key factor affecting both attractiveness and immunity is adiposity. Obesity contributes to an altered immune function and reduced immunocompetence because it is associated with changes in leucocyte counts, reduced antibody production, impaired wound healing, a higher risk of infections, and even a higher mortality rateOverall, evidence pertaining to links between the quality of the immune system and facial attractiveness is ambiguous. Many previous studies investigated only a limited number of relationships between variables and relied on indirect measures of immune system functioning. In Study 1, we therefore focused on the relationship between immune system reactivity and perceived facial attractiveness. To measure the reactivity of the immune system, we experimentally activated the immune system by vaccination against both viral  and bacterial (meningococcus) infections, because the two in conjunction should stimulate a wider range of components of the immune system than either would. We used differences in antibody levels before and after vaccination as a proxy for reactivity of the immune system. In Study 2, we investigated associations between immune system reactivity and perceived skin patch healthiness to examine human ability to judge characteristics from limited amount of information. Finally, in Study 3 we focused on the relationship between immune system reactivity and perceived healthiness of the face. Moreover, we measured testosterone and cortisol levels and recorded body composition, because all these factors have immunomodulatory properties and are linked to both perceived facial attractiveness and healthiness. We also measured facial skin colouration  to assess its role in attractiveness and healthiness judgements and its connection to the immunoreactivity.53, face, and voice as perceived by opposite-sex individuals. The present article focuses on associations between immune system reactivity and perceived facial attractiveness, healthiness, and skin healthiness. All procedures were conducted in accordance with the Helsinki Declaration and the study was approved by the Institutional Review Board of Charles University . Due to the nature of this study, we have collaborated with medical personnel. The study was preregistered prior to data analyses (https://osf.io/69zgc). Before entering the study, all participants were informed about its goals and expressed their consent with participation by signing an informed consent form.Data used for this study are part of a larger project which investigates possible associations between reactivity of the immune system and attractiveness of human body odour54).We have collected data from 21 men . Requirements for participating in the study were the following: age 18–40 years, good general health, no current use of any medication, non-smokers, and not being vaccinated against hepatitis A, B, or the meningococcus in the past 10 years  and leaflets at university halls of the Faculty of Science, Faculty of Humanities, and Faculty of Physical Education and Sports . Participants were vaccinated free of charge and received a reimbursement of 400 CZK (approx. €15) for participation in the whole project as a compensation for their time and potential inconvenience. Targets were the same for all studies described in the present article (Studies 1–3).55, that is, such values do not indicate currently ongoing infection. After the blood collection, the vaccines against hepatitis A/B (Twinrix) and meningococcus (Menveo) were administered. We selected vaccines against both viral and bacterial infections to stimulate different components of the immune system . The second blood collection and second photograph acquisition took place 14 days after vaccination, at a time point when one should expect the highest antibody response56. For the current investigation, only photographs taken before the vaccination were used. The last blood collection took place 30 days after vaccination, at a point when a second dose of vaccine against hepatitis (Twinrix) is recommended57. Vaccination and first blood collection were performed by a physician, while the remaining two blood samples were collected by phlebotomists at the Prevedig laboratory (https://www.prevedig.cz/) where all samples were subsequently analysed. The procedure and time of blood collections were standardised across participants. To avoid diurnal fluctuations58 sampling was conducted at 7–8 a.m. We measured and recorded body composition of the participants. Participants also completed questionnaires about their health status during the study and about possible factors that may have influenced their skin colour 60. This procedure took place on Q4 2017 to minimise possible effects of a suntan.One day before vaccination, we acquired standardised portrait photographs of the participants. On the day of the vaccination, each participant completed a questionnaire on their medical history and their general health status was examined by a physician to ensure they were eligible for application of the vaccines and not suffering from any current illness or infection. This was followed by the first blood collection (5 ml of venous blood) to assess the basal levels of antibodies (specific immunoglobulins G—IgG and immunoglobulins M—IgM) and C-reactive protein (CRP), which is a marker of inflammation. In none of the participants did the pre-vaccination CRP levels exceed 5.5 mg/l; values below this threshold are considered clinically normalNeisseria meningitis serogroups A, C, Y, and W-135). They can be administered together and are widely used in the Czech Republic. Both were applied intramuscularly, each in one arm.To induce an immune system response, we used the Twinrix Adult vaccine against hepatitis A/B and a Menveo vaccine against meningococcus  were measured by the Diasorin® Liaison—chemiluminescence immunoassay (CLIA), where a fully automated immunological analyser performs the full processing of samples. We used the corresponding Human S100 CLIA kits. This analysis is based on a radioimmunoassay, where the antigen and paramagnetic microparticle solid phase binds with fluorescent-labelled antibodies and after oxidation–reduction reaction, excessive energy is released in the form of photonsAntibodies against hepatitis B (Anti-Hbs) were measured based on the same principle as Anti-HAV. It turned out, however, that large percentage of targets either had high levels of Anti-Hbs at the baseline (N = 7) or did not respond to vaccination (N = 5). For this reason, the Anti-Hbs were excluded from further analyses.62. Sufficient response is at least 1:4 titres (the dilution of the serum where antibodies still react with the antigens) and ideally even higher63. As above, the final photometric measurement and evaluation were conducted by the analyser.Antibodies against the meningococcus (Anti-Mnk) were measured by the fully automated Diasorin® ETI-Max 3000—enzyme immunoassay (ELISA), one of the basic methods of determination of serum antibodies. The method is based on a reaction between an antigen on a special board and antibodies in the patient’s serum. Then secondary antibodies are added, which are specially labelled and bind to the primary antibodies with the antigen. A chromogenic substrate, which is added last, causes a colour response that is measured by spectrophotometer64. The final photometric measurement and evaluation were done by the automatised analyser.Total testosterone levels were measured by chemiluminescence (CLIA) in a fully automatised analyser Beckman Coulter DxI 800 Immunoassay System. The CLIA principle is described above. In this case, the energy is released by a reaction between testosterone, polyclonal anti-testosterone antibodies, and a tracerCortisol levels were measured by an electrochemiluminescence immunoassay method in a fully automatised analyser Beckman Coulter DxI 800 Immunoassay System. First, one incubates a sample in which specific anti-cortisol antibodies labelled with ruthenium chelate bind to cortisol. This complex is captured on the surface of an electrode where the electric charge causes a chemiluminescent emission of photons. The emitted light is measured by a spectrophotometer, but the measurement and evaluation are likewise done by the analyser.65.Acquisition of photographs took place at the Human Ethology perception lab in a purpose-built photographic booth in order to prevent potential changes in ambient illumination and colour reflections66 (Nikon AF-S NIKKOR 85 mm f/1.8G) into 14-bit uncompressed raw files (.NEF) and Adobe RGB colour space. The camera was mounted in a portrait orientation directly on a light stand that also carried a strobe light. A single 400Ws studio strobe (Menik MD-400Ws) was used and equipped with a white reflective umbrella light diffuser  mounted onto a 175 cm high light stand tilted 10° downwards toward the booth. Correct and uniform exposure across the entire scene was checked before each session with a digital light meter (Sekonic L-308S). Colour calibration was performed using X-Rite Color Checker Passport colour targets and a white balance patch photographed at the beginning of each session. For further details of the photo acquisition procedure, see Třebický et al.65.Portrait photographs were taken with a 24-megapixel full-frame  digital SLR camera Nikon D610 equipped with a 85 mm fixed focal length lensParticipants were photographed wearing provided white T-shirts and without any adornments or glasses. They had varying amount of facial hair ranging from clean-shaven to a full beard (in two participants), but most targets had a comparable style of short stubble. Participants were seated on a barstool 0.5 m from a plain white background. They were asked to sit straight with hands hanging freely alongside their bodies, look directly into the camera, and adopt a neutral expression. The camera  was positioned 125 cm from the participant and its height adjusted individually for each target to centre his head in the middle of the frame [distance between the camera and the participant was checked with a digital laser rangefinder (Bosch PLR 15)]. This setting of camera distance, focal length, and sensor size yielded a 35 × 53 cm field of view (23.85° angle of view).Image processing was carried out in Adobe Lightroom Classic CC (version 2017) and Adobe Photoshop CC 2015. We converted the images into DNG raw files and created DNG colour calibration profiles (using the X-Rite Color Checker Passport Lightroom plugin). Then we applied the profiles to all photographs. The calibrated images were exported into 16-bit Adobe RGB TIFF files in their actual size (35 × 53 cm) with a 168 PPI resolution. We manually checked the exposure (using the eye-drop tool on the background above the participants’ heads) and corrected the exposition on 85% value of every channel in the RGB colour space if necessary. Horizontal and vertical position of each participant in the image was adjusted using the Lightroom Transform tool . Then we batch-cropped to fit the heads on 27″ monitors in 1:1 size.In the next step, we removed any possible disturbing creases or shadows in the background. Finally, we converted the photographs into an sRGB colour space and exported them into an 8-bit JPEG format  for the rating.67.Facial skin colour was measured in vivo with a spectrophotometer Ocean Optics Flame-S with optical resolution of 2 nm using a standard D65 illuminant. Integrating Sphere ISP-R was used to spatially integrate the radiant flux in scatter transmission and diffuse reflectance sample measurements. The spectrophotometer was calibrated using the WS-1 Diffuse Reflectance Standard. All measurements were taken on three patches of targets’ faces  and expressed also in CIE L*a*b* colour space68. We measured the largest available area per stimulus while avoiding freckles, blemishes, and hair whenever possible. Facial skin colour values obtained from the spectrophotometer and from the facial photographs in our sample correlated positively . To facilitate a comparison with previous studies, we decided to use in further analyses in the main text facial skin colour measurements from the photographs. The results of analyses using spectrophotometer can be found in the Supplementary Materials—Tables We have also measured facial skin colour from the calibrated pre-vaccination facial photographs using ImageJ software (v 1.51) and Color Transformer 2 MatLab package. We measured the skin colour in CIE L*a*b* colour space and recorded the values for facial redness (a*), yellowness (b*), and lightness (L*) in three places of the face 69).We cropped skin patches from the obtained facial photographs in Adobe Photoshop CC 2015. The area of skin patches (89 × 89px) and location from which they were acquired (left cheek and forehead) were standardised while making sure that the resulting patch did not include any facial features , hair, or birthmarks. The resulting skin patches  were enlarged by 300% for subsequent presentation , oral invitations, and posters in university halls of the Faculty of Science, Faculty of Humanities, and the Faculty of Physical Education and Sport . In Study 1, facial photographs were rated by 88 females aged 18–40  during Q1 2018. The raters received a reimbursement of 200 CZK (app. €8) as a compensation for their participation in the whole project .In Study 2, the obtained photographs and skin patches were rated by 62 females aged 18–40  during Q1 2019. The raters received a reimbursement of 50 CZK (app. €2) as a compensation for their time.In Study 3, the photographs were rated by 66 females aged 18–40  during Q4 2019. They received a reimbursement of 150 CZK (app. €6) as a compensation for their participation in a larger rating session unrelated to the current investigation.None of the raters in the three studies used hormonal contraception.53. Exposure to a higher number of odour samples increases the risk of olfactory adaptation and can therefore affect rating, which is why the rating sessions were conducted on two separate days to accommodate a larger number of raters. From the total of 88 raters, 43 took part on the first day and 45 on the second day, which corresponds to the number of raters per photograph (depending on which day the photograph was presented). Randomly selected half of pre-vaccination samples was presented on the first rating day and half of post-vaccination photographs was presented on day two and vice versa. All raters were assessing photographs only once within a single day.Study 1 was part of a larger project that also included the rating of voice recordings and body odour samplesStudy 2 was carried out using same rating procedure to eliminate any possible effects of a different data collection design. Of the 62 raters, 32 rated the first half of the randomly selected stimuli and 30 the second half of the stimuli. For Study 3, the procedure was identical and of the 66 raters, 31 took part on the first data collection day, 35 on the second day.All rating (Study 1–3) took place in the Human Ethology perception lab under standardised conditions across all raters and rating days . The rating was conducted using Qualtrics survey suite  on two desktop computers of identical configuration with colour and brightness calibrated (by X-Rite i1Display Pro probe) LCD monitors  turned to a vertical position to accommodate life-sized facial images.70. Photographs were presented in randomised order.The raters were seated 115 cm from the screen with eyes at a height of 125 cm (measured from the floor to the outer corner of the eye). This is a height and distance comparable to that from which the portrait photographs were taken, whereby raters were positioned into the same centre of projection and eye level. This setup approximates the common interpersonal distanceIn Study 1, all facial photographs (N = 21) were rated for attractiveness on a 7-point verbally anchored scale. In Study 2, participants rated portrait photographs (N = 21) on a 7-point verbally anchored scale for attractiveness again to check the robustness of acquired ratings. They also rated skin patches from left cheek (N = 21) and forehead (N = 18) on a 7-point verbally anchored scale regarding their healthiness. Due to a low number of forehead patches (hair in the images), we use only cheek patches in analysis below. In Study 3, portrait photographs (N = 21) were rated on a 7-point verbally anchored scale regarding healthiness.After rendering their rating assessments (Study 1–3), raters completed a questionnaire about their basic demographics .To determine the consistency of raters’ assessments, we performed an intra-class correlation (ICC) analysis for each group rating the same set of samples using IBM SPPSS Statistics (v 23). All remaining statistical analyses were performed in jamovi (v 1.6.15).71. Further, we used the Spearman’s rank correlation to test the association between levels of antibodies and targets’ age.To explore relationships between variables, ratings of facial attractiveness from Study 1 and 2, and facial healthiness from Study 3, we used Spearman’s rank correlation coefficient because the data deviated from normal distribution. We set ρ ≥ 0.8 as a value at which we would consider the two variables highly correlated. In such case, only one of the variables would be used for subsequent analysesWe used a one-way analysis of variance (ANOVA) with Tukey post-hoc test to test for differences and Spearman’s rank correlation coefficient for strength of associations between separate colour measurements from the right and left cheek and the forehead.To examine the relationship between perceived facial attractiveness (Study 1), perceived skin patch healthiness (Study 2), perceived facial healthiness (Study 3), and differences in antibody levels (pre-vaccination subtracted from 30 days post-vaccination), we specified three separate linear mixed-effects models (LMMs) using the GAMLj module in jamovi. The rated characteristics  were entered as dependent variables, while differences in antibodies against hepatitis A (Anti-Hav) and meningococcus (Anti-Mnk) were entered as predictors. To control for variability in targets and raters, we entered the targets’ and raters’ IDs as random effects  + (1|ID_donor)). We employed analogous models to assess the relationships between the rated characteristics , steroid hormones levels, and the percentage of adipose tissue, and to assess the relationship between the rated characteristics  and forehead and cheek lightness, redness, and yellowness. To explore a possible relationship between targets’ age and perceived facial attractiveness and healthiness, we ran analogous separate linear mixed-effects models, with the rated characteristic entered as a dependent variable and age as the predictor.To test the association between differences in antibody levels (pre-vaccination and 30 days post-vaccination), basal levels of steroid hormones (testosterone and cortisol), and the percentage of adipose tissue, we employed general linear models (GLM) using the GAMLj jamovi module. In both models, we entered specific antibodies (Anti-HAV or Anti-Mnk) as dependent variables and steroid hormones and percentage of adipose tissue as predictors ). Analogous tests were carried out to investigate the relationship between antibody levels (pre- and 30 days post-vaccination) and forehead and cheek lightness, redness, and yellowness. For information about model residuals, see Supplementary Materials 72 to estimate observed power using the SimR package73 in R . Further, based on simulated data , we plotted Power curves showing the sensitivity to detect observed effects with α = 0.05. The results of observed power, Power curve plots, and the R script are available in the Supplementary Materials We performed a simulation-based power analysis for each fixed-effect predictor in our LMMsDescriptive statistics for targets’ basic demographic data, rated characteristics, differences between pre- and 30 days post-vaccination antibody levels, steroid hormone levels, the percentage of adipose tissue, and colour measurements are presented in Table We found high level of agreement between raters in all rated characteristics (ICC above 0.864). For further details, see Table p < 0.001). In all subsequent analyses, we therefore use attractiveness ratings from Study 1.Ratings of facial attractiveness collected in Study 1 and 2 were strongly positively and statistically significantly correlated . The value of ρ did not, however, reach the pre-set level of 0.8, and we therefore analyse perceived facial attractiveness and healthiness separately.Ratings of perceived facial attractiveness (Study 1) and perceived healthiness (Study 3) were also positively and statistically significantly correlated  did not show a statistically significant association . The relationship between perceived facial healthiness and targets’ age  was likewise not statistically significant . Further, we found no statistically significant relationship between antibody levels and targets’ age . In subsequent analyses, we therefore did not control for age.Linear mixed-effects model testing the relationship between targets’ age and perceived facial attractiveness (Rp < 0.001), redness , and yellowness  were strongly positively and statistically significantly associated. We thus continue to use only L* a* b* measures from the left cheek in further analyses because we presented the left cheek patches to participants in Study 2 for patch healthiness ratings.Left and right cheek measures of skin lightness , redness , and yellowness  from the left cheek and forehead were also positively and statistically significantly correlated. The ρs did not, however, reach the predefined level (0.8) and we therefore use the left cheek and forehead measures in further analyses separately. For further details, see Table Skin lightness  and statistically significantly less red (a*) and less yellow (b*) than skin on either cheek (for skin yellowness (b*), there was a statistically significant result for forehead and left cheek only). The two cheeks did not differ significantly in either L*, a*, or b* measures  was not predicted by levels of specific antibodies. For details, see Table Linear mixed-effects models show that perceived facial attractiveness . For detailed results, see Table  Neither redness, yellowness, nor lightness of the forehead or left cheek predicted elevations in any of the specific antibodies . For details, see Table Running a linear mixed-effects model, we found that neither the levels of cortisol or testosterone, nor the percentage of adipose tissue predicted perceived facial attractiveness  and Anti-Mnk . For detailed results, see Table In a GLM analysis, neither the levels of testosterone or cortisol, nor the percentage of adipose tissue predicted elevations in any of the specific antibodies Anti-HAV , forehead redness was the only statistically significant predictor with a negative slope ; see Table In a linear mixed-effects model testing the influence of skin colour on perceived facial attractiveness  was not predicted by levels of specific antibodies , cheek redness negatively predicted perceived cheek patch healthiness. For detailed information, see Table A linear mixed-effects model shows that perceived cheek patch healthiness  was not predicted by levels of specific antibodies , forehead redness negatively predicted  perceived facial healthiness  testing the association between cortisol, testosterone, adipose tissue percentage, and facial healthiness shows that only cortisol levels marginally negatively predicted  perceived facial healthiness. For details, see Table A linear mixed-effects model  and perceived attractiveness and healthiness. We found no statistically significant associations between experimentally elicited levels of antibodies against hepatitis A (Anti-HAV) or meningococcus (Anti-Mnk) and perceived facial attractiveness, healthiness, or healthiness of skin patches. Moreover, we observed no statistically significant associations between the levels of antibodies and testosterone, cortisol, or adipose tissue, which are all variables often associated with immune function. Adipose tissue and testosterone and cortisol levels also showed no connection with perceived facial attractiveness. Notably, we found a small negative effect of cortisol levels on perceived facial healthiness. Further, we found that higher forehead redness was perceived as less attractive and healthy when individuals assessed portrait photographs, and for cheek patches, higher cheek redness was perceived as less healthy. No systematic relationship was found between measures of facial skin colouration and Anti-HAV and Anti-Mnk antibodies.7 or elevated immune system response to vaccination against hepatitis B 9. Although the 95% confidence intervals of our results regarding the association between Anti-HAV, Anti-Mnk, and perceived facial attractiveness  do partially overlap with results of some studies that found significant relation between perceived facial attractiveness and hepatitis B antibodies levels after vaccination9, our results are more in line with the studies by Skrinda et al.11 and Rantala et al.10 who found no support for significant associations between hepatitis B antibody levels after vaccination and perceived attractiveness in men  and women , respectively. Overall, as noted at the outset, empirical evidence regarding an association between immunocompetence and facial attractiveness remains equivocal75.We examined possible relationships between immunoreactivity and facial attractiveness and healthiness because it has often been claimed that attractive traits are related to underlying qualities of individuals. Previous studies indeed reported a positive link between male facial attractiveness and either cytokine levels after stimulation with LPS 76. Moreover, we inspected both immunoreactivity and facial colouration. Unlike some previous studies50, we excluded hepatitis B antibodies from our analyses because several participants showed high levels of the relevant antibodies already in the baseline measurement, while others did not react to the vaccine.The strength of our study lies in using vaccines against both viral (hepatitis A and B) and bacterial (meningococcus) diseases. This way, we aimed to stimulate a wider array of immune system components because, for example, the advantage of heterozygotic individuals is the greatest when they fight against multiple pathogens at once77. Excessively strong (hypersensitivity) or inappropriate  immunity response is not beneficial and can ultimately negatively affect individual fitness. Moreover, by focusing solely on antibody levels, one can only arrive at generalised and limited information about the function of the immune system. Investigation of differences of the immune response in its humoral and cellular components and of the trade-offs between them might provide a more nuanced insight.In general, the use of vaccination to stimulate immunoreactivity has some limitations. For the purpose of this study, we treated a higher level of antibodies as a proxy to higher disease resistance. This is, however, something of a simplification because higher immunoreactivity is not always adaptive78 focused on innate immunity and measured salivary immune function  alongside oxidative stress and semen quality. Using principal component analyses, they obtained two factors: PC1—bacterial-killing capacity and overall bacterial immunity and PC2—bacterial suppression capacity and lysozyme activity. Contrary to expectations, no connection was found between the selected physiological measures of immune function, attractiveness  and number of sexual partners 78. Phalane et al.7, on the other hand, found a positive relationship between cytokine levels (after stimulation of the immune system with LPS)  and male facial attractiveness but not the CRP 7. Cai et al.79 employed as a marker of immune function salivary immunoglobulin A (IgA), which acts as a defence against microbial invasion. They found no connection between IgA and female facial attractiveness 79.Recent studies employed several methods of measuring the functioning of the immune system and arrived at rather diverse results, making our null results no exception. Foo et al.80. It has been demonstrated that people can assess health and attractiveness even from limited information such as skin patch and their ratings correspond to their ratings of the whole face69. In our study, we therefore used cheek skin patches to limit possible effects of confounding factors . We also investigated relationships between perceived healthiness of the skin patch and direct measures of immune system function. And yet, we found no associations between perceived skin healthiness and levels of specific antibodies.It has been proposed that facial skin provides information about the functioning of the immune system and about health40. Although the results of some studies do support the hypothesis of immunosuppressive effects of sex hormones81, the overall pattern in literature is rather mixed . It has been suggested that glucocorticoids contribute to this complex picture because they modulate immune system response as well as the expression of secondary sexual characteristics, and they may interact with testosterone45. In our study, we found no significant effects of either testosterone or cortisol on antibody levels. This finding is consistent with the results of Nowak et al.82, who found no influence of testosterone on the effectiveness of immune system using the influenza vaccine. In contrast, though, in vitro studies did find an immunosuppressive effect of testosterone on a spontaneous production of IgG in mononuclear cells of human peripheral blood84. Rantala et al.9, however, showed that the immune system’s reactivity was higher in males with higher testosterone levels who simultaneously exhibited lower cortisol levels and, moreover, these males were perceived as more attractive by women. In our study, we found none of the expected associations between testosterone and the rated characteristics. We found only a weak negative association between cortisol and perceived healthiness, but not attractiveness. Interestingly, another study showed a negative association between attractiveness  and cortisol levels45.Previous studies reported that testosterone and cortisol have an effect on both the functioning of the immune system and perceived facial attractiveness and healthiness, and might thus work as mediators between the functioning of the immune system and perceived facial characteristics. According to the hypothesis of immunocompetence handicap, androgens exert immunosuppressive effects and only high-quality individuals (including their immunity) can produce and maintain a high level of testosterone and afford the physiological costs of lowered immunosuppression45. This suggests that one should exercise caution when selecting specific characteristics to be rated for individual studies.Additionally, we found that the two scales of facial attractiveness and healthiness are positively correlated but the magnitude of this association is not strong enough to treat the two as interchangeable. It is thus possible that facial attractiveness and healthiness stand for two separate perceptual qualities. This idea finds further support both in the negative association between cortisol levels and perceived facial healthiness reported in our study and in the negative association between cortisol levels and perceived attractiveness in a study by Moore et al.48 Moreover, the faces of obese and overweight individuals are perceived as less attractive51. Adiposity thus seems to underlie the relationship between immune response and attractiveness50. In our study, we did not find any significant relationship between perceived facial attractiveness or healthiness and antibody levels or body fat percentage. One possible explanation might be that participants in our sample had a generally lower body fat percentage (mean = 17.5%): only two participants fell in the obese category with body fat percentage over 25% (threshold recommended by the American Council on Exercise). Our sample, where variability of body fat percentage was relatively low, may have been thus ill-suited to detecting the negative effect of increased adiposity. On the other hand, other studies detected a negative effect of higher weight (expressed by BMI) on immune function even within the range of average body weight variation85.It has been reported that higher adiposity contributes to reduced immunocompetence, and possibly impaired immune function accompanied by changes in leukocyte counts, lower antibody production, as well as worse wound healing and higher risk of infections7. Furthermore, yellower skin was preferred alongside lighter skin, but it is well possible that this preference for lighter skin is due to the yellow carotenoid colouration being more visible in lighter skin hues7. In our study, however, we found no statistically significant associations between skin yellowness and perceived characteristics. A number of other studies  employed manipulation of skin colour in photographs, while we used natural portrait images. This may have resulted in a lower variability in our sample, thus potentially reducing the likelihood of observing the effect. Still, we found that both perceived facial attractiveness and healthiness were negatively predicted by higher forehead redness and cheek skin healthiness was negatively predicted by higher cheek redness. Although higher redness has been previously linked to higher perceived attractiveness and health17, the relationship need not be linear: it is possible that some level of redness may affect perceived attractiveness positively, but above a certain threshold it has a negative effect on perceived attractiveness16. We propose that higher (forehead) redness levels might be perceptually linked to dermatoses, such as rosacea86, acne, or other imperfections which are generally perceived as less attractive80.A number of previous studies reported associations between skin colour, facial appearance, and immune response. South African men with a higher cytokine response to stimulation (induced by LPS) had yellower, more ‘carotenoid’ skin colour87, we used facial skin colour measurements from the cheek and the forehead separately, as majority of colour measurements between those areas differed and were only moderately associated. Cheeks and forehead differ in the amount of subcutaneous fat and therefore also in blood perfusion, which might account for slight differences in colouration. Accordingly, it has been found that the variation of colour in different parts of the face matters, whereby for instance periorbital luminance, cheek redness, and overall yellowness of the face predict perceived health88. To some extent, though, the differences in the skin colour of various parts of the face in our sample might be also due to the methods we used for acquisition of facial photographs from which we measured the values of facial colours. Our aim was to simulate naturally occurring daylight conditions with a diffuse strobe light positioned above the participant’s head pointing downwards. In this setup, though, the light source was positioned relatively close. Due to inverse-square law of loss of light over distance, it may have reflected on the forehead, causing it to appear brighter than the cheeks, and it may have produced highlights responsible for the observed colour differences between forehead and cheeks. Further, we tried to limit any potential effects of bright spots and light reflections by patting participants’ foreheads with napkins and we waited for some time before taking the photographs to avoid any skin redness caused by this process. Still, some brighter areas may have appeared and caused specular highlights, thus affecting the measurements.Unlike various studies which measured skin colour from both cheeks and the forehead and averaged them into one value for facial skin lightness, redness, or yellowness89), which resulted in wide confidence intervals of the effect sizes and a low power to observe the reported effects (in most cases below 50%). Based on our Power curve analysis, even a sample size of 100 targets would not yield a higher power .Aside from the limitations discussed above, the main limitation of the present study is the small sample size of targets  as well as anti-vaccination biases which may have discouraged some individuals from participation10 and our results are based only on a male sample. Future investigations should thus include both men and women as targets and raters to better understand the complex relations between attractiveness and immunity and its role in intersexual selection.Previous research has also suggested that immunoreactivity and cues to various aspects of immune system functioning may differ between the sexesWe investigated the relationship between functioning of the immune system and perceived facial attractiveness, healthiness, skin patch healthiness, and potential influence of skin colour. We employed measurements of antibodies after application of two different vaccines as markers of reactivity of the immune system and recorded the levels of steroid hormones (cortisol and testosterone) as well as the percentage of adipose tissue due to their immunomodulatory properties and connection to facial attractiveness. We found no significant relationships between reactivity of the immune system and perceived facial attractiveness, healthiness, and skin patch healthiness. We did, however, observe a small negative effect of cortisol on perceived facial healthiness. Moreover, steroid hormones and adipose tissue showed no relationship to either the immune response after vaccination or skin colouration. Finally, higher forehead redness from portrait photographs was perceived as both less attractive and healthy and higher cheek redness from skin patches was perceived as less healthy. Our results thus suggest that facial attractiveness and healthiness provide a limited amount of cues to immune system functioning and perceived characteristics seem to be related only to certain hormone levels and facial colour.Despite some limitations, we believe that our study is a valuable contribution to research on the role of visual cues in assessments of functioning of the immune systems of individuals, and that it can serve as an entry for future meta-analysis aimed at disentangling the conflicting results of various existing studies. Future studies might also investigate the activation of different components of the immune system, such as humoral and cellular immunity, and focus on acquiring larger samples.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.


HIV rapid testing services is one among key interventions in the controlling of HIV/AIDS. Despite availability of quality standards, the quality of HIV rapid testing services remains questionable since non-laboratory testers are allowed to conduct testing while they are not specialized in providing testing services.To evaluate the compliance to the quality standards of HIV rapid testing services provided by non-laboratory testers in Makete District, TanzaniaAn explanatory descriptive study employing quantitative approach of data collection was used. An observation of 23 non-laboratory testers performing HIV rapid tests, observation of HIV testing points and documents review was done in 23 testing points to collect data. Data were analyzed using a programmed excel sheet and a three-point scale was used to determine the level of compliance to quality standards.Analysis shows that out of 23 testing points visited, the level of compliance to quality standards was lower for 22 (95.6%) testing points and moderate in 1 (4.4%) testing point. None of the testing point was highly complied to quality standards for HIV rapid testing services.The quality of HIV rapid testing services provided by non-laboratory testers is below the established quality standards for HIV rapid testing services. HIV rapid testing services is among key interventions in the controlling of HIV/AIDS since it provides chances to access HIV care, treatment, prevention, and support services.5Several studies have been conducted to evaluate the quality of HIV rapid testing services. An auditing study conducted in Caribbean and African countries, which involved 968 HIV testing points demonstrated that 20% of the testing points scored level 0; 46% of the testing points scored level 1; 31% of the testing points scored level 2; while 3% of the testing points scored level 3 and none of the testing point that had high level of compliance to quality standards such that none of the testing points scored level 4.6Furthermore a study conducted in 102 private health facilities in Kampala Uganda to evaluate the quality of HIV rapid testing services demonstrated that the quality of HIV rapid testing services is below the recommended quality standards.7Tanzania has also adopted the WHO quality standards for the implementation of HIV rapid testing services.The study was conducted at Makete District Council in Njombe Region, Southern Tanzania. HIV Records for 2016/2017 show that Njombe Region has the highest HIV prevalence (11.4%) in Tanzania.Purposive sampling technique was used to select both public and private health facilities where by all HIV rapid testing points were studied and non-laboratory testers who provided services on the day of the study were observed when performing actual HIV rapid tests.An on-site checklist for document review and observation adopted from WHO by the Tanzania Ministry of Health was used to collect quantitative data. The main areas of observations included: personnel training and certification; physical facility; safety; pretesting phase; testing phase; documentation and records as well as external quality assessment.Ethical clearance to conduct this study was obtained from Research and Ethics Committee (REC) of Muhimbili University of Health and Allied Sciences (MUHAS). Permission to conduct the study was requested from the District Medical Officer of Makete District and officer in charges of the respective health facilities. Study participants were informed about the purpose of the study, procedures, risk and benefits for them to participate in the study. Informed written consent was obtained from each study participant after agreeing to participate in the study.Data were collected manually and entered into excel sheets and they were cleaned and validated. All descriptive data were analyzed using programmed excel sheets and data were presented in frequency tables of numbers, percentages  and level of compliance to quality standards for HIV rapid testing service. Summation of each response of the open ended question in each quality standard was used to determine the average compliance for each quality standard. A three-point scae  was used to determine the level of compliance to all quality standards for HIV rapid testing services for each testing points whereas testing points scored level 4 (score of 90% or higher) were defined as highly complied to quality standards; level 3 (score of 80% – 89%) were defined as moderately complied to quality standards; level 2 (60% – 79%), level 1 (40% – 59%) and level 0 (less than 40%) were defined as lower complied to quality standards.Most of the HIV testers (34.8%) worked on wards, Reproductive and Child Health section (21.7%) and Care and Treatment Centre (13%). The majority of the testers (52%) were from the district level health facilities while about 22% of them were from dispensaries. Majority of the testers (78%) worked in the government health facilities and the remaining 22% worked in private health facilities. Most of testers were women (78.3%) while 21.7% were men. All testers (100%) had ordinary level of secondary education whereas among them 69.6% testers had certificate level of education and 30.4% had diploma level of education. Only one study participant (4.4%) was clinical officer, 30.4% were enrolled nurses and 34.8% were Medical attendants while 30.4% were assistant nursing officers.Evaluation results show that out of 23 testing points visited, the level of compliance to the quality standard was lower for 22 (95.6%) testing points. Two (2) testing points (8.7%) scored level zero, 13 (56.5%) testing points scored level one and 7 (30.4%) scored level two. The level of compliance to HIV rapid testing quality standards was moderate in 1 (4.4%) testing point. None of the testing point had high level of compliance to the HIV rapid testing quality standards, meaning that no testing point attained level four. Average compliance for each quality standard was also determined by observing how non-laboratory testers and testing points operations comply with such standards. These standards include: personnel training and certification; physical facility, safety, pretesting phase, testing phase, documentation and records as well as external quality assessment. The average compliance to personnel training and certification quality standards was 15% whereas: only 9 (39%) out of 23 non laboratory testers that provided HIV rapid testing services on the day of the study received comprehensive training on HIV rapid testing using the nationally approved curriculum; only 2 (9%) non-laboratory testers were trained on external quality assessment (EQA)/proficiency testing (PT) process; only 5 (22%) non-laboratory testers were trained on safety and waste management procedures and practices. No any non - laboratory tester has been certified or licensed through a national certification program to perform HIV rapid testing services.Average compliance to physical facility quality standards was 65% whereas: 16 (66%) testing points have designated area for providing HIV rapid testing services; 11 (48%) testing points were clean and organized for testing; 22 (96%) of testing points had sufficient lighting; none of the testing points store test kits in temperature-controlled environment and there was no device to monitor temperature in all visited testing points and 18 (78%) testing points had sufficient and secure storage space for test kits and other testing consumables.Average compliance to safety standards was 56% whereas: only 2 (9%) testing points had SOPs to manage spills of blood and other body fluids; only 4 (17%) testing points had SOPs to address accidental exposure to potentially infectious body fluids through a needle stick injury, splash or other sharps injury; all 23 (100%) testing points had personal protective equipment (PPE) and testers properly used PPEs through the testing process; 13 (57%) testing points had hand-washing facilities such as water and liquid soap. Only 6 (26%) testing points handled properly waste materials such as sharps, infectious and non-infectious waste and only 13 (56%) testing points disinfect working area with the appropriate disinfectant such as chlorine.The average compliance to pre testing phase standards was 63% whereas: only 9 (39%) testing points had national guidelines for HIV testing services; all 23 (100%) testing points use national algorithm and approved HIV rapid kits; expired HIV rapid test kits such as uni-gold were available in 9 (39.1%) testing points; only 4 (17%) testing points had a standard process for stock management; only 4 (17%) testing points had posted job aides on client sample collection; 19 (82%) testing points had sufficient supplies for client sample collection and in only 9 (39%) testing points, client's identifiers were recorded in the HIV testing register and on test devices as per national guidelinesThe average compliance for testing phase standards was 53% whereas: only 5 (54%) testing points had SOPs on HIV testing procedures and posted at the testing area; timers were available and used routinely in only 1 (4.3%) testing point; sample collection devices such as capillary tube, loop, disposable and pipettes were used accurately in 22 (95%); testing procedures such as correct use of buffer and reading of results on time were adequately followed in 16 (70%) testing points and positive and negative quality control specimens were routinely used in 13 (57%) testing points. Retest for verification was done in 4 (67%) health facilities out of 6 health facilities visited.Average compliance to documentation and records standards was 92% whereas: 20 (87%) testing points had standardized register for documentation, which contain key elements in the registers such as client demographics, kit names, lot numbers, expiration dates and tester name. The final HIV results were recorded correctly in 16 (70%) testing points; client documents and records were securely kept throughout all phases of the testing process in 22 (95%) testing points.Average compliance to External Quality Assessment was 40% whereas: 11 (48%) testing points were enrolled in Proficiency Testing (PT) program; out of 11 testing points enrolled into PT program, only 4 (36%) testing points implement corrective action in case of unsatisfactory PT results; 21 (91%) testing points received periodic supervisory visit and written supervision report was available in only 1 (5%) testing point.This study has shown that the level of compliance to the quality standards for HIV rapid testing is below the recommended standards. Majority of observed HIV testing points scored level one and none of the testing point was highly complied to quality standards, meaning that no testing points scored level four. The results of this study are similar with the study conducted in Kampala Uganda to assess the quality of HIV counseling and testing services, which demonstrated that the quality of HIV testing services is below the recommended standards.13In this study, the highest average compliance score was seen on documentation and records followed by physical facility that included availability of conducive designated areas for provision of HIV rapid testing services and the lowest average compliance score was seen on personnel training and certification, followed by External Quality Assessment, testing phase, safety and pre testing phase. In addition, this study revealed that availability of new National HIV register that require all key elements to be documented in one register has contributed to improved documentation and record keeping for different data hence results into higher score for documentation and records quality standards. Furthermore, this study shows that most of the non-laboratory testers did not receive comprehensive training to perform HIV rapid testing services and none of them were certified/licensed to perform HIV rapid test through the National Certification Program. This situation has resulted into the lower score for the personnel training and certification quality standards, which was below the recommended quality standards for HIV rapid testing services, which requires that services should be provided by testers who received comprehensive training as well as evaluated for their competence and certified to provide testing services.7In this study we observed low compliance to the quality standards for External Quality Assessment (EQA) and about half of the testing points were enrolled into proficiency testing program and most of the testers were not trained to perform samples for proficiency testing. A study conducted in South Africa to evaluate the quality management system for HIV rapid testing services reported similar findings indicating that the compliance to EQA standards was low and only two clinics out of eleven clinics were enrolled into proficiency testing program. 6In this study, non-adherence to testing procedures including the use of small volume of buffer and non-adherence on time to read result whereby timers were not used during testing were observed. Similar findings were reported in a study conducted in Zambia National Reference Hospital in which the use of inadequate volume of specimen and use of incorrect volume of buffer leading into poor quality of test results was reported.11Our study has shown that a few testing points had guidelines for HIV testing services and Standard Operating Procedures (SOPs). These guidelines and SOPs are important documents for providing guidance and procedures needed to be followed during testing hence lack of guidelines and SOPs compromises the quality of HIV rapid testing services.19Some of the testing points were temporarily closed during data collection period due to lack of trained testers to provide service, and probably such testing points could have useful information regarding the practice of implementation of HIV rapid testing services in studied facilities. Furthermore, due to resource constrains only six facilities were studied, which might have limited the data collected. However, we believe that this study provides valuable insights regarding the quality of HIV rapid testing services provided by non-laboratory testers in the rural setting district in Tanzania.The quality of HIV rapid testing services provided by non-laboratory testers in Makete District is below the established quality standards set for provision of HIV rapid testing services. Most of the non-laboratory testers were not trained, evaluated for their competencies and were not certified or licensed to provide HIV rapid testing services. Some of HIV rapid testing points do not have Guidelines and SOPs that guide provision of HIV rapid testing services, which compromises the quality of HIV testing services. The Ministry of Health and Management of the health facilities should invest more efforts in training and strengthening of certification of non-laboratory testers, increasing enrolment of testing points into proficiency testing and strengthening supervision of testing points in order to improve compliance to quality standards.


Here, we assessed the effects of imbalanced NKCC1 and KCC2 on γ-aminobutyric acidergic (GABAergic) neurotransmission in certain brain regions involved in human focal cortical dysplasia (FCD).Altered expression patterns of NaWe sought to map a micro-macro neuronal network to better understand the epileptogenesis mechanism. In patients with FCD, we resected cortical tissue from the seizure the onset zone (SOZ) and the non-seizure onset zone (non-SOZ) inside the epileptogenic zone (EZ). Additionally, we resected non-epileptic neocortical tissue from the patients with mesial temporal lobe epilepsy (MTLE) as control. All of tissues were analyzed using perforated patch recordings. NKCC1 and KCC2 co-transporters expression and distribution were analyzed by immunohistochemistry and western blotting.Results revealed that depolarized GABAergic signals were observed in pyramidal neurons in the SOZ and non-SOZ groups compared with the control group. The total number of pyramidal neurons showing GABAergic spontaneous postsynaptic currents was 11/14, 7/17, and 0/12 in the SOZ, non-SOZ, and control groups, respectively. The depolarizing GABAergic response was significantly dampened by the specific NKCC1 inhibitor bumetanide (BUM). Patients with FCD exhibited higher expression and internalized distribution of KCC2, particularly in the SOZ group.Our results provide evidence of a potential neurocircuit underpinning SOZ epileptogenesis and non-SOZ seizure susceptibility. Imbalanced function of NKCC1 and KCC2 may affect chloride ion homeostasis in neurons and alter GABAergic inhibitory action, thereby contributing to epileptogenesis in FCDs. Maintaining chloride ion homeostasis in the neurons may represent a new avenue for the development of novel anti-seizure medications (ASMs). Focal cortical dysplasia (FCD) is a common cause of refractory epilepsy . HistoloA receptor agonists; this remains a clinical challenge. Recent studies have demonstrated that the Na+-K+-2Cl– (NKCC1) and K+-Cl– (KCC2) cotransporters alter GABAergic inhibitory function by regulating intracellular chloride concentration ([Cl–]i) (GABA) and prominent GABAA receptor (GABAAR)-mediated spontaneous and evoked depolarizing responses in pediatric patients with severe FCD exhibiting epilepsy. –]i caused by whole-cell recordings or the lack of intracellular recordings.Epilepsy results from an imbalance between excitatory and inhibitory synaptic transmission in the brain . Previou([Cl–]i) . Histolo([Cl–]i) . In addi([Cl–]i) . This suAt the macroscopic level, based on electrocorticography (ECoG) data This study sought to compare the detailed electrophysiological characterization of GABAergic neurotransmission in pyramidal neurons in the SOZ and non-SOZ inside the EZ of patients with FCD with non-epileptic temporal neocortex and to correlate these features with the patterns of NKCC1 and KCC2. We probed whether depolarizing GABAergic action caused by the imbalanced function of NKCC1 and KCC2 cotransporters would perturb chloride ion homeostasis in neurons and contribute to the generation and propagation of epilepsy in patients with FCD.In this study, 30 patients with intractable epilepsy were enrolled at Xuanwu Hospital of Capital Medical University from June 2018 to December 2019. Following a standardized preoperative evaluation, all patients underwent surgery to remove the EZ. Neocortical tissues were obtained from 20 patients with FCD and epilepsy. In addition, 10 patients with mesial temporal lobe epilepsy (MTLE) were chosen as the control group because of their relatively non-epileptic neocortical tissue, as suggested by previous studies . The temEach patient recruited in the study underwent standardized preoperative evaluation procedures to delineate the EZ and locate the SOZ, including detailed clinical history, neurological examinations, video-electroencephalographic recordings (vEEG), neuropsychological examinations, and neuroimaging studies, etc. Neuroimaging studies include high-resolution magnetic resonance imaging , magnetoencephalography, and 18-fluorodeoxyglucose positron emission tomography (FDG-PET). As mentioned above, Given that the brain tissue is taken during surgery, the surgeon cannot cut off the blood supply in order to keep the brain tissue alive before collecting the brain tissue. When we collected the first two samples (SOZ and non-SOZ) from the first patient, it was shown that collecting two brain tissues at the same time would cause more serious bleeding which did inevitable damages to the patient. Therefore, from the second patient, we adopted a double-blind and random sampling method, and each patient randomly collected one piece of brain tissue. All patients with FCD were confirmed by a postoperative histological examination . In summ2 and 5% CO2) cutting solution containing 220 mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2, and 10 mM D-glucose , and transferred to our electrophysiological laboratory within 5–10 min. Subsequently, meninges and blood clots were gently removed, and 400 μm neocortical transverse slices were cut using a vibratome  and incubated in oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 2 MgCl2, and 10 D-glucose  at room temperature for at least 1 h before recording.After being obtained in the operating room, neocortical tissues were immediately placed into an ice-cooled and oxygenated . Gramicidin was stocked at an initial concentration of 16 mg/ml and then diluted with pipette solution to reach a final concentration of 80 μg/ml. The electrode tip and barrel were backfilled with pipette solution and gramicidin-containing pipette solution, respectively. Following the formation of a tight seal and after the series resistance (Rs) decreased and stabilized at 30–110 MΩ, we started recording. Recordings were ended when a rapid decrease in Rs and a “leak-like” current were observed, indicating that the cell had entered the conventional whole-cell configuration by rupture of the membrane seal. The resistance and capacitance of the pipette have been compensated. Liquid junction potential was not corrected.Following incubation, the brain slices were submerged in a recording chamber perfused with oxygenated ACSF at a rate of 2–2.5 mL/min at room temperature. Perforated patch-clamp recordings were performed on pyramidal neurons in the neocortex and viewed under an infrared differential interference contrast microscope with a Nikon 40 water immersion lens . Recordings were performed in layers III and IV in the non-SOZ and control groups. In the SOZ group, in which the lamina was not clearly defined, layers III and IV were generally located according to the non-SOZ lamina in the middle region of the dysplastic cortex . Pyramidm), membrane conductance, and firing properties to identify pyramidal neurons in the current-clamp mode. Next, we recorded spontaneous excitatory postsynaptic currents (sEPSCs) for 3 min at a holding potential of −70 mV in the voltage-clamp mode. Subsequently, we acquired pharmacologically isolated GABAergic sPSCs by applying 6-cyano-7-nitroquinoxaline-2,3-dione  and DL-2-amino-5-phosphonopentanoic acid  to block α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. BUM (10 μM) was then added to assess its effects on GABAergic sPSCs. The sEPSCs were automatically detected at amplitude adjusted above the root 2 mean square noise level using Clampfit 10.5 . Cumulative amplitudes were calculated by summating amplitudes of all events within 180 s periods.We first recorded the resting membrane potential  from a pipette resting 10 –20 μm above the slice at the position of the recorded soma. The system enabled quick fluid changes with a speed of 1 ml/min and a diameter of about 20 μm for its tip. Voltage ramps −80 to + 10 mV over 100 ms applied from a holding potential of −70 mV in the absence and presence of GABA were used to determine EGABA. The membrane voltage at which the current traces, obtained in the presence and absence of GABA, crossed was measured as the apparent EGABA. Then, voltage ramps were repeated following the addition of BUM (10 μM). The membrane voltage at which the current traces, obtained in the presence and absence of BUM, crossed was measured as the apparent EBUM. In addition, the driving force for GABA production (DFGABA = EGABA–Vm) was measured.In this study, to probe the EThe brain tissue was fixed in 10% formalin and embedded in paraffin. Paraffin-embedded tissues were sectioned at 6 μm and heated in a microwave oven (60°C for 60 min). The sections were thereafter deparaffinized in xylene and rehydrated sequentially in graded concentrations of alcohol. Sections from the SOZ, non-SOZ, and control groups were probed with an anti-KCC2 antibody  overnight at 4°C. The slides were then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Immunolabeling was visualized using the avidin-biotin conjugation method and 3,3′-diaminobenzidine. The sections were next counterstained with hematoxylin.Brain slices were post-fixed with 10% formalin, washed three times with PBS, incubated for 30 min with 0.3% Triton, and then blocked with 1% goat serum for 1 h to avoid binding of non-specific antibodies. Sections were then incubated at 4°C overnight with the following primary antibodies: anti-NKCC1 , anti-NeuN . Secondary antibodies conjugated to Alexa Fluor 488 or 594  were applied for 1.5 h at room temperature. Slides were mounted with Fluoroshield containing DAPI  and observed with a confocal microscope .Total protein was quantified using a BCA Protein Assay Kit . Samples were electrophoresed on 5 and 10% Tris–HCl gels, transferred onto polyvinylidene difluoride membranes, and incubated with primary antibodies . GAPDH  was used as the reference protein. Horseradish peroxidase-conjugated secondary antibodies  were used to bind the primary antibodies. The bands were visualized using a two-channel infrared (IR) scanner . Densitometric analysis of the protein signals was carried out using the ImageJ software . The target protein levels were normalized to internal control.t-test or one-way ANOVA with post-hoc tests. Results were expressed as mean ± standard error of the mean (SEM), with significance set at p < 0.05. For the skewed distribution, analysis was carried out using the non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparisons or Mann–Whitney U-test. Results were expressed as medians and interquartile range (IQR), with significance set at p < 0.05.All data distributions were assessed with the Shapiro–Wilk test. For the normal distribution, data were analyzed using Student’s n = 9), temporal (n = 10), or posterior quadrants (n = 1).First, we analyzed the clinical characteristics of the patients included in this study. No significant differences in the mean duration of epilepsy were observed across the groups. Briefly, the male/female ratio of patients with FCD exhibiting epilepsy enrolled in the study was 1:1, the mean duration of epilepsy being 13.5 ± 1.8 years and the average age at surgery being 22.9 ± 2.1 years old. The EZ in the FCD was localized in frontal , input resistance (RN), or membrane time constant  and non-SOZ group . However, no significant difference was observed between the SOZ and non-SOZ groups. The median amplitude of sEPSCs on pyramidal neurons was not significantly different among the three groups. The decay time was smaller in the non-SOZ group  than in the control group  and the SOZ group . In addition, no significant differences in the sEPSC rise time were observed across the groups  with picrotoxin at a holding potential close to RMP in patients with tuberous sclerosis . We normalized GABAAR-mediated sPSCs before and after the application of BUM (10 μM). BUM reduced GABAAR-mediated sPSCs frequency  and cumulative amplitude  in FCD patients  than in the non-SOZ . There was no significant difference in the frequency of GABAAR-mediated sPSCs , the amplitude of GABAAR-mediated sPSCs  and the cumulative amplitude of GABAAR-mediated sPSCs  in the pyramidal neurons of between SOZ and non-SOZ groups  was significantly shifted to more positive values compared to that in the control group  and non-SOZ group  was more positive than RMP  in the SOZ group. In contrast, no significant differences were observed between EGABA and RMP in the non-SOZ  and control groups . DFGABA was significantly higher in the SOZ group than in the control group , but EGABA shifted to more negative values in the SOZ group (−52.9 vs. −75.0 mV) (Entersect . The ave75.0 mV) .In addition to the electrophysiological results indicated significant depolarizing GABAergic signaling in neurons in the SOZ group, we assessed the neuronal expression of NKCC1 and KCC2 in the three groups . As prevDiffuse neuropil staining for KCC2 was observed in the FCD cortical tissue as well as in the control temporal neocortex. The neurons in the SOZ showed that KCC2 was internalized into the cells and that its distribution on the cell membrane was significantly reduced. However, there was no obvious KCC2 cell internalization in the non-SOZ and control groups. Histological analyses revealed cytoplasmic KCC2 accumulation in neurons in the SOZ group, whereas cytoplasmic KCC2 staining was absent in the non-SOZ and control groups . A westeGABA in the SOZ than in the non-SOZ, which may explain the macroscopic differences between the SOZ and non-SOZ in patients with FCD. In addition, we revealed that depolarizing GABAergic neurotransmission in FCD patients, which can be reversed by BUM, is related to the imbalanced function of NKCC1 and KCC2 in the SOZ. This is the first comparative study of depolarizing GABAergic signals in the SOZ, non-SOZ, and non-epileptic cortical regions. Our findings provide important evidence of a potential neurocircuit underlying SOZ epileptogenesis and non-SOZ seizure susceptibility. These results also point to a possible mechanism for epileptogenesis in FCD at the cellular and microcircuit levels, highlighting a novel therapeutic avenue for patients with FCD and epilepsy.xIn this study, we assessed the electrophysiological characteristics and depolarizing synaptic activity of pyramidal neurons in patients with FCD and epilepsy. In particular, we probed the effects of imbalanced function of NKCC1 and KCC2 on GABAergic signaling in pyramidal neurons inside the SOZ, non-SOZ within the EZ of the patients with FCD, and non-epileptic neocortex in patients with MTLE (control). We sought to establish a relationship between macroscopic epileptogenesis and microscopic cell-to-cell communication to better understand the epileptogenic network of FCD through a micro-macro neuro-electrophysiology lens. We observed depolarizing GABAergic signaling in pyramidal neurons located in the SOZ and non-SOZ of the FCD, but not in the control group. Although there is no significant difference between SOZ and non-SOZ in terms of frequency and intensity of depolarizing GABAergic responses at this microscopic level, however, there were more neurons with depolarizing GABAergic responses and higher EAccurate delineation of the EZ is critical to determining which patients with refractory epilepsy are appropriate candidates for surgical intervention . Althoug– transporters and highest EGABA compared to the subiculum and hippocampal proper in patients with MTLE, which indicates relatively balanced NKCC1 and KCC2 level  temporal neocortical tissue lies far from the EZ and is not characterized by any obvious structural aberration ; (2) temC2 level ; and (3)in vitro. In contrast, mainly inhibitory GABA actions was shown in the immature hippocampus and neocortex in vivo (in vitro preparations is currently highly debated (A activation and GABAAR-mediated synaptic transmission changes from inhibitory to excitatory effects caused by altered NKCC1 and KCC2 (AR-mediated postsynaptic responses, depolarized EGABA in pyramidal neurons at the single-cell level, and corresponding changes in NKCC1 and KCC2 in the SOZ and non-SOZ of patients with FCD exhibiting epilepsy, when compared to those obtained from non-epileptic human temporal neocortex from patients with MTLE. Moreover, we found several differences in GABA activity between the SOZ and non-SOZ groups in the EZ of patients with FCD, highlighting the importance of depolarizing GABA within a seizure network.The recruitment of glutamatergic neurons is a fundamental component of seizures . As 80%  in vivo . It is t debated . Furtherand KCC2 . In our in vivo, which has been shown to impair neuronal network formation (–, which are modulated by the extrusion of Cl– by KCC2. Thus, the loss of KCC2 function results in the neuronal accumulation of Cl–, shifting the direction of ion flow through GABA receptors from hyperpolarizing to depolarizing (In our study, consistent with previous studies, we observed that KCC2 expression was downregulated and internalized in the EZ of patients with FCD, especially in the SOZ . Althougormation . Moreovelarizing .– inward. We found that the imbalanced function of NKCC1 and KCC2 in the EZ resulted in depolarization of GABA receptor function and may underpin the hyperexcitability of FCD brain regions. Compared with the control group, SOZ and non-SOZ of patients with FCD showed depolarizing GABA response on pyramidal neurons. Although there were no significant differences in terms of the frequency and intensity of the depolarizing GABA response across the SOZ and non-SOZ groups, fewer neurons with depolarizing GABAergic responses and higher EGABA were shown in the SOZ than in the non-SOZ of FCD patients. The migration of seizures from one side to the other requires the presence of dynamic depolarizing GABA in the network (The change of GABA function in the SOZ can strongly affect the microcircuit connection and neuronal network, as well as activate and silence individual neurons. In support of the critical role of KCC2 in postnatal GABAergic inhibition,  network . Therefo network . AlthougAR-mediated postsynaptic responses in cortical slices of patients with FCD and epilepsy at the individual cell level. However, the use of BUM as a potential new antiepileptic drug warrant further examination. Future studies should examine BUM derivatives as novel ASMs. In addition to NKCC1, an inhibitor of KCC2 should be explored for its effect on depolarizing GABA responses.In our study, we focused on the depolarizing GABA synaptic activity caused by the imbalance of NKCC1 and KCC2 and aimed to build a micro-macro neuronal network to determine the relationship between depolarizing GABA and epileptogenesis in FCD. We observed depolarizing GABAergic signaling among pyramidal neurons in both the SOZ and non-SOZ of the patients with FCD, with a higher number of responsive neurons in the SOZ group. Thus, we speculated that abnormal GABAergic activity might influence epileptogenesis in the SOZ group and seizure susceptibility in the non-SOZ group. The microscopic recordings within areas of macroscopic evaluation and definition, that is, SOZ and non-SOZ, also pointed to the importance and potential of mapping epileptic activities at multiple scales. In addition, given the effects of BUM, the maintenance of chloride homeostasis in neurons could represent an alternative avenue for developing ASMs. Previous studies have reported that BUM exerted significant seizure control effects in adult patients with temporal lobe epilepsy . In our This study had several inevitable limitations. First, although we found the specific NKCC1 inhibitor BUM significantly reduced the depolarizing GABAergic response, it is hard to figure out why BUM functions as a result from functional upregulation of NKCC1 or decreased expression of KCC2 in FCD. To solve this issue, we think that the single-channel recording and the specific and available blocker of KCC2 is in urgent need. Second, it remained difficult to obtain SOZ and non-SOZ from the same patient with FCD to avoid excess injury and protect important functional brain areas during surgery. Finally, the size of our cohort was relatively small, and we did not take a deeper look into the differences across variable FCD subtypes and neuronal subgroups. In the future, a larger cohort of patients should be recruited to assess the potentially different GABAergic functions across FCD subtypes and other types of neurons, such as interneurons.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Xuanwu Hospital, Capital Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.GZ and XFY: supervision and conceptualization. RL: methodology and writing – original draft. YX, HZ, JW, HLL, and LC: writing – review and editing. DL: visualization. TY, XMY, CX, YP, and LZ: provide clinical patient data, tissue specimens, and pathological examination. HHL: review and edit the manuscript. All authors contributed to the article and approved the submitted version.


The benign aggressive tumour known as a giant cell tumour of bone (GCTB) frequently affects the knee bones. Patients suffering from GCTB present with pain, swelling, joint effusion, loss of ability to bear weight on the involved extremity and a restriction in the range of motion of the afflicted joint may also exist, depending on the tumour's size. GCTB makes up 20% of benign skeletal tumours and 5% of all primary bone tumours. Although it has an equal distribution of the sexes, the majority reveal a higher frequency among women. Eighty per cent of GCTB instances were recorded in patients between the ages of 20 and 50 during the third decade. The femur, tibia and radius are where GCTB is most frequently discovered. Lesions can be rated using the Campanacci grading method based on the plain radiograph's results. Plain radiography, CT and MRI are used to diagnose the tumour. Surgery is the only curative treatment which is determined by the Campanacci grade and the tumour's location. Recurrence of GCTB is observed in about 25% of patients, with curettage being associated with rates as high as 50%. We evaluated the GCTB-related articles and summarised the developments in diagnosis, treatment and reducing risk of recurrence. Giant cell tumour of bone (GCTB), first described in 1818, is a benign primary bone tumour marked by multinuclear giant cells and has a high propensity to return locally after treatment . NeoplasIt is uncommon for GCT to involve the metatarsal bone in patients with underdeveloped skeletons . It is aEpidemiologyGCT makes up 20% of benign skeletal tumours and 5% of all primary bone tumours. Southern India and China, where GCT accounts for 20% of all primary bone tumours, have an especially high prevalence. Although some studies have claimed an equal distribution of the sexes, the majority reveal a higher frequency among women. Eighty per cent of GCT affects patients between the ages of 20 and 50 during the third decade when the prevalence of the condition increases. Less than 3% of cases include children under the age of 14, and only 13% involve patients over the age of 50. Most lesions (75%-90%) form in long bones, with the knee accounting for the bulk of cases (50%-65%) . Paget dSiteAlthough GCTB can develop anywhere in the skeleton, it usually manifests as an extension of the metaphysis into the epiphysis in long bones . The disGrading systemThe tumour often shows up as an irregular region of bone loss on conventional radiography. In cases of a higher grade, the cortex may thin and perforate. Lesions can be rated using the Campanacci grading method based on the plain radiograph's results. Lesions of Grade I are limited to the bone. Grade II lesions are those that feature an enlargement of the cortex but no perforation and Grade III lesions are those that have soft tissue extension and cortical perforation . The difSymptomsPain is the most typical GCTB-presenting symptom. Additional symptoms include swelling, joint effusion, and a restriction in the range of motion of the afflicted joint may also exist, depending on the tumour's size. Some symptoms, including neurological symptoms in GCTB of the axial skeleton, are specific to the location of the tumour. When the cortex of weight-bearing bones thins, patients may even present with pathological fractures .ImagingA physical examination is done after getting a patient's medical history. Plain radiography, computed tomography (CT), and magnetic resonance imaging (MRI) are used to assess the main tumour. While plain radiography and CT are helpful for assessing the tumour's bone component, MRI is better suited for evaluating the tumour's intramedullary and soft tissue expansion. Finding pulmonary metastases, which may exist in a very tiny proportion of patients, requires the use of a chest radiograph. To find more skeletal illness areas, bone scans are helpful . The "doAmong all medical imaging techniques, MRI is the most crucial since it enables disease diagnosis, disease severity evaluation, and therapy planning. In up to 67% of cases, the preoperative MRI not only identifies the condition but also matches the postoperative findings . For theDiagnosisGiant cells are found in many benign and malignant bone lesions, hence it is important to correlate clinical, imaging, and pathologic data when diagnosing GCTs in order to rule out other lesions with a similar histologic pattern . In GCTBTreatmentAt this time, proper surgery is the only curative treatment for GCTB. In general, the sort of surgery that would be suitable is determined by the Campanacci grade and the tumour's location . Curettage and adjuvant therapy are preferred for Campanacci grade 1 or 2 tumours (which do not extend beyond the cortex), but tumours with soft tissue extension (Campanacci grade 3) are treated with extensive local excision due to their high risk of recurrence . AlthougRiskWith the use of local adjuvant therapy, the high risk of recurrence associated with intralesional surgery can be reduced to about 30%. The Scandinavian Sarcoma Group demonstrated in their retrospective analysis that the likelihood of recurrence was decreased from 61 to 22% when cement rather than bone graft was used to fill the surgical void . HoweverHistologyGCTB has a friable look and a dark brown to reddish colour. The histological appearance of GCTB traditionally exhibits a stroma of mononuclear spindle cells and monocytes in the foreground and several big multinucleated giant cells in the background . Neoplasa. Primary malignant GCTB, pre-denosumab: Undifferentiated pleomorphic sarcoma is indicated by the proliferation of atypical spindle and pleomorphic cells developing in fascicles; GCTB is indicated by the proliferation of ovoid to spindle bland-appearing cells with scattered reactive multinucleated osteoclast-like large cells.b. Secondary malignant GCTB, pre-denosumab: GCTB-like histological characteristics coexisted with an unusual spindle cell proliferation that invaded between the host bone trabeculae, which was a sign of a high-grade, undifferentiated spindle cell sarcoma .According to available data, benign GCT of bone has three different cell types:Type I cells have the ability to multiply, resemble interstitial fibroblasts, and produce collagen. The tumour component of GCT is probably this cell population. The mesenchymal stem cells from which type I cells may have been formed share some characteristics with type I cells; however, type I cells also have characteristics that may indicate an early differentiation of mesenchymal stem cells into osteoblasts.Type II cells can be recruited from the peripheral bloodstream and are also interstitial, but they resemble the monocyte/macrophage family. By fusing with one another or by nuclear multiplication without cell division, these cells are regarded to be the forerunners of the multinucleated giant cells. Giant cells lack the monocyte/macrophage lineage surface receptors that are expressed by type II cells.The multinucleated large cells are type III cells. They morphologically resemble osteoclasts and share many of their traits. They have bone resorption enzymes such as type II carbonic anhydrase and tartrate-resistant acid phosphatase. The majority of the unique osteoclast antigens are shown by GCT large cells. There are calcitonin and vitronectin cell receptors, and matrix metalloproteinases (MMPs) are expressed .Tumour markersSerum acid phosphatase has been studied for its potential utility in GCT as a tumour marker. According to one study, half of the cases had high acid phosphatase levels, while the other half had levels that were normal. Acid phosphatase concentrations were higher in those with larger GCT lesions. However, postoperatively, serum acid phosphatase levels significantly dropped in both populations. In spite of these findings, acid phosphatase is not a reliable screening indicator for sufficient resection or local recurrence. The tumour marker creatine kinase isoenzyme BB (CK-BB) is also being studied in GCT. In order to regenerate adenosine triphosphate, this enzyme catalyses the reversible transfer of phosphate from phosphocreatine to adenosine phosphate. It is hardly ever discovered in the serum of healthy people. Patients with brain traumas, stomach cancer, prostate cancer, and particular forms of osteopetrosis have seen it most frequently. Patients with osteosarcoma, aneurysmal bone cysts, malignant fibrous histiocytomas, or common osteolytic entities do not have increased levels. Patients with GCT have high CK-BB levels at the time of diagnosis, and two weeks following excision, their CK-BB levels recover to normal. In spite of these findings, CK-BB does not serve as a reliable screening indicator for sufficient resection or local recurrence .Latest advancementDenosumab may be administered as neoadjuvant chemotherapy in GCTB patients where surgical resection is the preferred main treatment option in order to facilitate tumour excision. To achieve a greater tumour resection margin, pre-operative denosumab is administered to inhibit tumour osteolysis, raise bone mineral density, increase marginal sclerosis, and reconstitute the articular surface . AdditioThe article provides more clarity about GCTB which is a benign aggressive tumour. The Campanacci grading method is the most frequently mentioned classification method. Major imaging technique like plain radiography, MRI and CT role in the diagnosis of GCTB has also been explained. Due to the development of these imaging techniques, it has become easy for doctors to diagnose GCTB. The article also explained different treatment procedures that are required to be followed based on the Campanacci grading system and the tumour's location. The risk factors involved in its treatment and chances of recurrence after treatment have also been assessed. The role of denosumab is also explained. Types of cells found in benign GCTB and histological characteristics of cancer in GCTB have also been discussed in this article.